The following includes information that may be useful in understanding various aspects and embodiments of the present disclosure. It is not an admission that any of the information provided herein is prior art, or relevant, to the presently described or claimed inventions, or that any publication or document that is specifically or implicitly referenced is prior art.
Noted for their biomimetic properties, hydrogels are used for biomedical applications, such as drug delivery, stem cell therapy and regeneration medicine and tissue engineering. Hydrogels are also favored in cell culture platforms due it their tissue-like softness and desired water content.
Traditional hydrogels made up of either synthetic polymers or natural biomolecules often serve as passive scaffolds for molecular or cellular species, which render these materials unable to fully recapitulate the dynamic signaling involved in biological processes, such as cell/tissue development. Thus, there is a need to design stimuli-responsive, dynamic hydrogels that can accommodate or mimic the complexity of biological systems.
A type of dynamically tunable hydrogel is a photoresponsive hydrogel. Photoresponsive hydrogels utilize light as a tool to control molecules or cell behavior with high spatiotemporal precision and little invasiveness. Through advancement of synthetic chemistry, progress has been made in making photoresponsive hydrogels with dynamically tunable properties. Through a combination of orthogonal click reactions and photochemistry, some of these synthetic hydrogels can be mechanically and chemically patterned in situ by light while being used for 3D cell culturing, and diverse photoactive chemical moieties have also been incorporated into synthetic hydrogels to create photoresponsive devices for controlled therapeutic release.
Assembling genetically engineered proteins into molecular networks represents an alternative strategy to make hydrogels with well-controlled properties. Although natural evolution has led to numerous functional protein domains that can sense and respond to a variety of environmental stimuli, such as light, oxidative stress, pH, small molecules, metal ions, etc., such ecological diversity has yet to be fully tapped to develop responsive biomaterials with dynamically tunable properties.
Cell culture is typically carried out by seeding a suitable medium with cells of a population to be expanded. Certain adherent cell types, such as human embryonic stem cells (hESC) and induced pluripotent cells (iPC's), are more effectively cultured by providing a surface upon which the cells can adhere to and proliferate. After adhesion and proliferation, the cultured cells need to be harvested and therefore released from the surface. Release of the cells is typically promoted by techniques such as mechanical scraping, chemical or enzymatic treatment, sonication, or a combination thereof.
The inventors have recognized that common cell release techniques can present a number of disadvantages. For example, mechanical scraping can damage the cells, and it is often not suitable for use in confined spaces such as small diameter wells or with three dimensional structures. The use of biological (e.g., protease), or thermal methods (e.g., lowering temperature below LCST) can present a few problems including inefficient release, cell damage or death and/or present a risk of introducing impurities into the cultured cells. For example, a common agent such as trypsin is known to promote deterioration of cell function. Furthermore, certain cells can be particularly adherent to a given substrate and need to be subjected to forcing conditions to promote their release, the effect of which results in a degree of cell damage.
As stated above, hydrogels, such as protein hydrogels, are used in cell culture platforms since they closely mimic native extracellular matrix, which is not only due to their identical nature of the chemical compositions (poly-(amino acid)), but also due to their similar built-in functional moieties (e.g., RGD cell adhesion site and matrix metalloproteinase cleavage site) that assist in cell adhesion or migration. As a result, cell growth, proliferation and differentiation can be controlled on such cell culture platforms including hydrogels.
However, even with the use of present hydrogels in cell culture platforms, there is still a need to improve cell release techniques to maximize harvesting cultured cells in an efficient manner while minimizing damage to and/or loss of the cultured cells.
Additionally, there is a need to improve synthesis techniques for stimuli-responsive “smart” protein-based hydrogels since it is a major challenge to assemble complex globular proteins into supramolecular architectures efficiently while preserving their function.